Journal of Dental Sciences 17 (2022) 802e810

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Original Article

Glycogen synthase kinase-3b inhibitor

promotes the migration and osteogenic
differentiation of rat dental pulp stem cells
via the b-catenin/PI3K/Akt signaling pathway
Huilan Xie*, Yi Lin, Fang Fang

Department of Stomatology, Fujian Provincial Hospital, Fuzhou, Fujian, China

Received 31 August 2021; Final revision received 26 September 2021

Available online 16 October 2021

KEYWORDS Abstract Background/purpose: Glycogen synthase kinase-3b (GSK3b) inhibitor enhances

Glycogen synthase bone formation, while dental pulp stem cells (DPSC) are potentially used to repair bone de-
kinase-3b inhibitor; fects. The present study aimed to investigate the effect of AR-A014418 (AR, a specific glycogen
Dental pulp stem synthase kinase-3b inhibitor) on the migration and osteogenic differentiation of rat-derived
cells; dental pulp stem cells (rDPSCs), and further explore the underlying mechanism.
Migration; Materials and methods: rDPSCs were isolated from rats, and then cultured with different con-
Osteogenic centrations of AR with or without LY294002 (a PI3K inhibitor). Then, cell viability, migration,
differentiation; osteogenic differentiation, and the involvement of PI3K pathway were detected by CCK-8
PI3K assay, Transwell assay, Alizarin Red S Staining, Alkaline phosphatase (ALP) assay, Western blot,
and RT-PCR, respectively.
Results: Our present study demonstrated that AR of various concentrations (1 mM, 2.5 mM, and
5 mM) not only promoted the rDPSC proliferation and migration, but also increased calcium
deposition, the activity of alkaline phosphatase (ALP), and levels of osteogenic markers
(RUNX2, OPN, OCN, and OSX) in rDPSCs. It was also found that the administration of AR resulted
in an increase in the expression level of p-GSK3b (Ser), b-catenin, p-PI3K, and p-Akt, and a
reduction in p-GSK3b (Tyr216). Furthermore, PI3K inhibitor LY294002 abrogated the enhanced
cell migration and osteogenic differentiation of rDPSCs induced by AR.
Conclusion: Our results provide evidence that AR significantly promotes migration and osteo-
genic differentiation of rDPSCs by activating b-catenin/PI3K/Akt signaling pathway.
ª 2021 Association for Dental Sciences of the Republic of China. Publishing services by Elsevier
B.V. This is an open access article under the CC BY license (http://creativecommons.org/
licenses/by/4.0/).

* Corresponding author. Department of Stomatology, Fujian Provincial Hospital, No. 134, East Street, Gulou District, Fuzhou, 350001,
Fujian, China.
E-mail address: carrie_hui37@163.com (H. Xie).

https://doi.org/10.1016/j.jds.2021.09.035
1991-7902/ª 2021 Association for Dental Sciences of the Republic of China. Publishing services by Elsevier B.V. This is an open access article under
the CC BY license (http://creativecommons.org/licenses/by/4.0/).
 Journal of Dental Sciences 17 (2022) 802e810
Introduction
As all known, stem cells can self-renew and differentiate
into other cell types. In the last decade, mesenchymal stem
cells (MSCs) have shown great potential in the field of
regenerative medicine due to their capacities of self-
renewal and multilineage differentiation.1,2 MSCs could be
isolated from numerous tissues including adipose tissues,3
bone marrow,4 skeletal muscle,5 and dental tissues.6 In
2004, Seo et al.7 initially isolated and identified dental pulp
stem cells (DPSCs) from the human pulp tissues. Since
DPSCs not only display a MSC-like property but also can be
conveniently harvested, they are considered promising
stem cell sources for clinical use.8 Extensive basic studies
proved the potent potential of DPSCs in the regeneration of
bone.9,10 Additionally, a previous clinical study confirmed
the safety and therapeutic effects of DPSCs in treating se-
vere periodontal defects.11 It is undoubted that the oste-
ogenic differentiation capacity of DPSCs is crucial for bone
regeneration. Besides, the migration of endogenous or
exogenous DPSCs to bone injury or loss sites is also signifi-
cant for bone regeneration.12 Therefore, enhancement of
osteogenic differentiation and migration of DPSCs is an
important avenue in improving the therapeutic efficacy of
DPSC-based therapy.
In recent years, increasing studies have found that
some small molecules show osteogenic function via
diverse signaling mechanisms.13e15 Given that it is easy to
synthesize small molecules, which are non-immunogenic
and cost-effective, it seems an attractive choice for
directing osteogenic differentiation and migration of
PDLSCs. Glycogen synthase kinase-3 (GSK3) is a
serineethreonine protein kinase containing two isoforms
(GSK3a and GSK3b), which is an important component of
various key signaling pathways, including insulin, mTOR,
NF-kB, and Wnt/b-catenin.16 Many studies have impli-
cated the Wnt signaling pathway in the lineage differen-
tiation and migration of MSCs.17,18 It is well known that
GSK3b inhibitors can activate the Wnt signaling pathway
by decreasing the degradation of b-catenin. Hence, GSK3b
inhibitors may provide a new therapeutic strategy for
DPSC-based bone regeneration. In the past decades, a
number of small molecule GSK3b inhibitors have been
identified;19e21 however, more than half of the published
inhibitors not only target GSK3b, but also affect cyclin-
dependent kinase 2 (cdk2) or cdk5.22 In 2003, Bhat
et al.23 identified an amino thiazole in a high-throughput
biochemical screening using purified recombinant GSK3b,
which was named as AR-A014418 (AR). AR can inhibit
GSK3b activity in an ATP-competitive way, with high
specificity for GSK3b as it does not affect the activity of
other 26 protein kinases, especially cdk2 and cdk5
(IC50 > 100 mM) that are inhibited by the majority of GSK3
inhibitors.23 Several studies reported that AR had potent
activities against diverse types of cancer with no adverse
effects in rodents.24,25 However, to our knowledge, the
803
role of AR in migration and osteogenic differentiation of
DPSCs has been rarely studied before.
The aim of this study is to investigate the effects of
GSK3b inhibitors, AR, on migration and differentiation of
rat DPSCs (rDPSCs) into osteoblasts in vitro, and prelimi-
narily explore the underlying molecular mechanisms.
Materials and methods
rDPSCs isolation and characterization
SpragueeDawley rats (3 months, SPF grade) were pur-
chased from Guangdong Medical Laboratory Center
(Guangzhou, China), and rDPSCs were isolated from the
dental pulp of incisors of rats, as Alksne et al.26 previously
described. The isolated rDPSCs were cultured in DMEM
supplemented with 10% FBS and 1% penicillin/streptomycin
at 37
C in a 5% CO2 incubator. The third rDPSC passage was
subjected to characterization, which included identifica-
tion of the cell surface markers and multidirectional dif-
ferentiation of rDPSCs. Every procedure was approved by
the Animal Care and Use Committee of Fujian Provincial
Hospital.
Osteogenic and adipogenic differentiation
For osteogenic differentiation, 5  104 rDPSCs were main-
tained in cultured media containing 10 mM b-glycerol
phosphate, 50 mg/mL L-ascorbic acid, 10 nM dexametha-
sone, and 2 mM glutamine for 7 or 14 days. During the
cultivation, the media were renewed three times every
week.
For adipogenic differentiation, 5  104 rDPSCs were
maintained in cultured media containing 5 mg/mL insulin,
0.5 mM 3-isobutyl-1-methylxanthine, and 1 mM dexameth-
asone for 14 days. During the cultivation, the media were
renewed every other day.
Alizarin red S (ARS) staining
ARS staining was conducted to confirm osteogenic differ-
entiation and assess calcium-rich deposits in rDPSCs. In
brief, after incubated with osteogenic media for 14 days,
rDPSCs were fixed with 4% paraformaldehyde (PFA) for
15 min and subsequently washed with PBS. 2% ARS solution
was added and incubated for 15 min. Thereafter, the
stained rDPSCs were observed under an optical microscope.
For quantitative evaluation, ARS was dissolved in 5%
perchloric acid by measuring absorbance at 562 nm under a
microplate spectrophotometer.
Oil red O staining
As a fat-soluble diazo dye that can stain lipid droplets in
cells, Oil Red O is usually utilized to confirm the adipogenic
 H. Xie, Y. Lin and F. Fang
differentiation of MSCs. Briefly, rDPSCs were fixed with 4%
PFA, followed by staining with Oil Red O solution in iso-
propanol for 15 min. Finally, differentiated oil red O-posi-
tive rDPSCs were observed and imaged under a microscope.
Western blot
rDPSCs were lysed in RIPA buffer (Abcam, Cambridge, MA,
USA) on ice to extract total protein. The total protein was
separated by SDS-PAGE gels and electrophoretically trans-
ferred onto PVDF membranes, which were subsequently
blocked in skimmed milk (12%) for 2 h. The membranes
were incubated overnight at 4
C with primary antibodies,
and subsequently rinsed three times before incubation with
the secondary antibody. Finally, protein bands were visu-
alized using ECL Substrate Kit (Abcam), and quantified using
Image J software (Bethesda, MD, USA). The information of
antibodies used in this study was described in
Supplementary Table S1.
CCK-8 assay
rDPSCs were cultured in media containing different con-
centrations (0, 1, 2.5, 5, 10, and 20 mM) of AR (Sigma-
eAldrich; A3230) for 1 day, 3 days, 5 days, and 7 days,
respectively. At the indicated time points, a CCK-8 reagent
(10%, v/v) was added to each well. Finally, the absorbance
at 450 nm was measured under a microplate reader (Bio-
Rad, Hercules, CA, USA) after additional 2-h incubation.
Transwell assay
Briefly, 700 mL cultured media were added to the bottom
chamber. Then, 200 mL rDPSCs suspension (serum-free) with
different treatments was added to the top chamber with a
filter membrane (8 mm pores), followed by 24-h incubation.
The cells which migrated to the bottom chamber were fixed
and stained. They were then photographed under a
microscope.
Alkaline phosphatase (ALP) assay
After incubated with osteogenic media for 7 days, the ALP
activity of rDPSCs was determined using colorimetric Alka-
line Phosphatase Assay Kit (Abcam). In short, rDPSCs were
incubated with ALP substrate for 15 min in the dark. Then,
the reaction was stopped by 2 M NaOH. The absorbance of
the product was detected at 405 nm under a microplate
reader.
RT-PCR
Total RNA was extracted from rDPSCs by using TRIzol reagent
(Life Technologies, Carlsbad, CA, USA). Thereafter, RT-PCR
analysis was conducted as described previously27 with the
following primer sequences: RUNX2 (F: 5-‘GATAACCTG-
GATGCCGTCGTG-30 , R: 50 -CAGCCTAGCCAGTCGGATTTG -30 ),
804
OPN (F: 5-‘CAGGTTCACCCCATTCTCC-30 , R: 50 -AAAT-
TATCCGGGCGTGGT-30 ), OCN (F: 5-‘CAGCGTTATGAGATCAA-
GATGACCA-30 , R: 50 -AGTGATGTGCAAGAGTCCATCCTG-30 ),
OSX (F: 5-‘GCTTTTCTGTGGCAAGAGGTTC-30 , R: 50 -
CTGATGTTTGCTCAAGTGGTCG-30 GADPH (F: 5-‘GAGAAG-
TATGACAACAGCCTC-30 , R: 50 -ATGGACTGTGGTCATGAGTC-
30 ). The subsequent quantification was carried out using the
QuantiTect SYBR Green PCR Kit (Toyobo, Osaka, Japan) on a
7500 Real-Time PCR System (Applied Biosystems, Förster,
CA, USA). GAPDH was selected as an internal control for
normalization in this study.
Statistical analysis
All data were expressed as mean  standard deviation (SD)
of three independent experiments. The Student’s t-test
was used for comparison between two groups. For multiple-
group condition, one-way ANOVA was performed with
Bonferroni’s method. P < 0.05 indicated significant
difference.
Results
rDPSC characterization
rDPSCs were isolated from incisors of rats, of which surface
antigens were characterized using Western blot. Compared
with non-DPSCs, CD29, CD44, CD90, and CD105 were obvi-
ously up-regulated, while CD34 and CD45 were down-
regulated in the isolated rDPSCs (Fig. 1A). ARS staining
and Oil red O staining respectively confirmed the role of
rDPSCs in osteogenic differentiation and adipogenic dif-
ferentiation (Fig. 1B). All these data suggested successful
isolation of rDPSCs.
AR enhances migration and osteogenic
differentiation of rDPSCs
Using the CCK-8 kit, the effect of AR (0, 1, 2.5, 5, 10, and
20 mM) on rDPSCs proliferation was observed (Fig. 2A). On
day 1 and day 3, there was no significant discrepancy in
rDPSCs proliferation between groups. After 5-day and 7-day
incubation, both 10 and 20 mM of AR suppressed prolifera-
tion of rDPSCs. Notably, after 5-day and 7-day incubation,
15 mM, 2.55 mM and 5 mM of AR enhanced proliferation of
rDPSCs. Hence, 15 mM, 2.55 mM and 5 mM of AR were
selected to investigate the effect of AR on rDPSCs migration
and osteogenic differentiation. As shown in Fig. 2B, the
migratory capacity of rDPSCs was promoted by AR in a
concentration-dependent way.
The biological role of AR in osteogenic differentiation of
rDPSCs was also explored using ALP assay, and ARS staining,
and the expression of osteogenic markers (RUNX2, OPN,
OCN, and OSX) was determined. The results showed that
the ALP activity of rDPSCs in 2.5 mM and 5 mM groups was
significantly higher than that in the control group after
osteogenic induction for 7 days (Fig. 2C). Similar to the
 Journal of Dental Sciences 17 (2022) 802e810
Figure 1 Characterization of rDPSCs. (A) Identification of stem cell markers in rDPSCs using Western blot. Normal fibroblasts
(NFs) without stemness were used as control. (B) Osteogenic differentiation (top) and adipogenic differentiation (bottom) of
rDPSCs detected by ARS and Oil Red O staining, respectively.
result of the ALP assay, the ARS staining showed there was
no significant difference between the control and 1 mM
groups (Fig. 2D). Besides, the deposition of calcium induced
by osteogenic media in rDPSCs was significantly increased
by 2.5 mM and 5 mM of AR (Fig. 2D). Moreover, both 2.5 and
5 mM of AR significantly elevated the expression of RUNX2,
OPN, OCN, and OSX in rDPSCs at mRNA and protein levels
(Fig. 2E and F). Taken together, our results indicated that
AR plays a positive role in migration and osteogenic dif-
ferentiation of rDPSCs.
AR promoted migration and osteogenic
differentiation of rDPSCs via the Wnt/b-catenin/
PI3K/Akt signaling
To preliminarily explore the potential mechanisms of AR
acting in migration and osteogenic differentiation, the ef-
fect of AR on the activity of Wnt/b-catenin, PI3K/Akt,
ERK1/2, and JNK signaling pathways in rDPSCs was investi-
gated. The results were depicted in Fig. 3. Compared with
the control group, AR treatment decreased the phosphor-
ylation of GSK3b at Tyr216 along with an increase in the
expression of b-catenin. Besides, the phosphorylation of
GSK3b at Ser9 was increased by the treatment of 5 mM AR.
These data suggested that AR could reduce the activation
of GSK3b, thereby increasing the expression of b-catenin to
activate Wnt/b-catenin signaling. In addition, the treat-
ment of AR also increased the phosphorylation of both PI3K
and Akt in a concentration-dependent way. The
805
phosphorylation of ERK1/2 was obviously increased when
the concentration of AR reached 5 mM. However, there was
no obvious difference in the phosphorylation of JNK among
all groups. Collectively, our results suggested that the Wnt/
b-catenin/PI3K/Akt signaling was involved in the effect of
AR on migration and osteogenic differentiation of rDPSCs.
Inhibiting PI3K reversed the effect of AR on
migration and osteogenic differentiation of rDPSCs
To confirm the role of PI3K/Akt signaling in the function of
AR in rDPSCs, rDPSCs were pretreated with LY294002
(15 mM), a specific PI3K inhibitor, for 1 h before AR treat-
ment (5 mM). LY294002 significantly weakened the migra-
tion ability of AR-treated rDPSCs (Fig. 4A). ALP assay and
ARS staining indicated that LY294002 effectively reversed
the increased osteogenic differentiation induced by AR
(Fig. 4B and C). Additionally, LY294002 also significantly
decreased the expressions of osteogenic markers in AR-
treated rDPSCs at both mRNA and protein levels (Fig. 4D
and E). We further investigated the effect of LY294002
interference on Wnt/b-catenin/PI3K/Akt signaling in AR-
treated rDPSCs. As expected, the treatment of LY294002
noticeably decreased expression of p-PI3K and p-Akt in AR-
treated rDPSCs. In the meantime, LY294002 also effectively
suppressed the decrease of p-GSK3b (Tyr216) and increase
of p-GSK3b (Ser9) and b-catenin induced by AR treatment.
In short, these data suggested that PI3K/Akt signaling
pathway was involved in the role of AR in migration and
osteogenic differentiation of rDPSCs.
 H. Xie, Y. Lin and F. Fang

Figure 2 The effect of AR-A014418 on cell proliferation, migration, and osteogenic differentiation of rDPSCs. (A) The cell
viability of rDPSCs determined by CCK-8 assay after 1-, 3-, 5-, and 7-day treatment. (B) The migration of rDPSCs detected by
Transwell assay. (C) The ALP activity of rDPSCs. (D) The calcium deposits in rDPSCs visualized by using ARS staining. The expressions
of osteogenic markers (RUNX2, OPN, OCN and OSX) were detected by (E) RT-PCR and (F) Western blot, respectively. **P < 0.01 and
***P < 0.005, versus control.

Discussion promoted the cell viability. Based on the results, a con-

DPSCs are an attractive source of MSCs for regenerative
medicine due to their relative accessibility and lack of
ethical controversy.28,29 It has been demonstrated that
DPSCs display greater proliferative potential than bone
MSCs.30 During the application of DPSCs for bone regener-
ation, it is required for DPSCs to expand and migrate to the
bone damaged/defect area and differentiate into osteo-
blasts. Hence, it is important to explore a strategy for
promoting DPSCs migration and osteogenic differentiation
in DPSC-based therapy. In this study, it was demonstrated
that AR, an inhibitor specific to GSK3b, may promote cell
migration and osteogenic differentiation via specifically
activating Wnt/b-catenin/PI3K/Akt signaling pathways in
rDPSCs.
In an attempt to determine the ideal concentration
range of AR on the cell proliferation of rDPSCs, a CCK-8
assay was carried out on rDPSCs after treated with diverse
concentrations of AR. In our study, after 7 days of incuba-
tion, AR suppressed cell viability of rDPSCs at a concen-
tration of 10 up to 20 mM, while no more than 5 mM
centration range of 1e5 mM was chosen for the subsequent
experiments. Thereafter, our data demonstrated that the
migratory ability of hDPSCs was enhanced by 2.5 and 5 mM
of AR. Previous studies indicated that GSK3 inhibitors
potentially promoted osteogenic differentiation of several
MSCs.31e33 Similar to the literature, our study showed that
the indicated concentrations of AR enhanced ALP activity
and calcium deposition. Moreover, both the mRNA and
protein expressions of RUNX2, OPN, OCN, and OSX were up-
regulated in a concentration-dependent way. RUNX2 is a
crucial transcription factor involved in osteoblast differ-
entiation, which regulates expression of many
osteogenesis-related genes.34 Takahashi35 indicated that
the overexpression of RUNX2 induced differentiation of
preadipocytes into functional osteoblasts. RUNX2 enhances
the expression of OSX,36 thereby inducing the expression of
OCN and OPN.37 These data collectively revealed that AR
promoted migration and osteogenic differentiation of
rDPSCs.
It is well known that Wnt/b-catenin signaling is involved
in tissue generation, regeneration, as well as self-

806
 Journal of Dental Sciences 17 (2022) 802e810
Figure 3 The effect of AR-A014418 on the Wnt/b-catenin, PI3K/Akt, ERK1/2 and JNK signaling pathways in rDPSCs. **P < 0.01
and ***P < 0.005, versus control.
renewal.38 Given that GSK3b is one of the key components
of Wnt/b-catenin signaling, we assessed the effect of AR on
the activity of the Wnt/b-catenin pathway. The activity of
GSK3b is down-regulated by phosphorylation at the Ser9
site, while up-regulated by phosphorylation at the Tyr216
site.16 The inactivation of GSK3b might lead to a decrease
in the degradation of b-catenin, thereby activating Wnt/b-
catenin signaling. Previous studies demonstrated that the
treatment of AR leads to a significant reduction in GSK-3b
phosphorylation at Tyr216 and regulates diverse biological
processes, such as cell growth,39 apoptosis,40 and sur-
vival.41 Besides, AR increases GSK-3b phosphorylation at
Ser9 when promoting osteogenic differentiation in human
adipose-derived MSCs.31 A similar result was obtained in the
present study. Once the concentration of AR reached 5 mM,
the Ser9 phosphorylation of GSK3b was significantly
enhanced, but 1 mM and 2.5 mM of AR did not affect Ser9
phosphorylation of GSK3b. Meanwhile, the administration
of AR resulted in an obvious decrease in the Tyr216 phos-
phorylation of GSK3b in a dose-dependent manner. Mean-
while, as expected, the protein level of b-catenin was
elevated in rDPSCs after treated with AR. These data
revealed that the activation of Wnt/b-catenin signaling
might be involved in the role of AR in rDPSCs migration and
osteogenic differentiation.
807
Moreover, our data also indicated that AR could increase
the activity of the PI3K/Akt pathway in a dose-dependent
manner, but displayed no obvious effect on JNK signaling in
rDPSCs. Our study also revealed that the activity of the
ERK1/2 in rDPSCs was obviously elevated only when the
concentration of AR reached 5 mM. This suggested that a
high dose of AR might further induce the activation of
ERK1/2 in rDPSCs. Since our results demonstrated that AR
promoted migration and osteogenic differentiation of
rDPSCs once its concentration reached 2.5 mM, the subse-
quent experiments merely focused on the role of the PI3K/
Akt pathway in the function of AR. Akt is an important
member of the protein kinase family, and it is at the center
of the signaling pathway, which is a biological signal
transduction pathway initiated by PI3K and can be acti-
vated by many stresses. Multiple studies implicated the
PI3K/Akt signaling pathway in cell proliferation and dif-
ferentiation.42,43 Accumulating evidence has demonstrated
that this pathway is involved in osteogenic differentia-
tion.44,45 Besides, a recent study has revealed that the
migration of DPSCs is associated with the PI3K/Akt
signaling.46 In our study, LY294002 was used to inhibit PI3K/
Akt signaling, and it was found that the treatment of
LY294002 reversed the enhanced migration and osteogenic
differentiation induced by AR. GSK3b is an important
 H. Xie, Y. Lin and F. Fang

Figure 4 PI3K inhibition by LY294002 blocked the promoting role of AR-A014418 in migration and osteogenic differentiation
of rDPSCs. (A) The migration of rDPSCs detected by Transwell assay. (B) The ALP activity of rDPSCs. (C) The calcium deposits in
rDPSCs visualized by using ARS staining. (D) The mRNA expressions of RUNX2, OPN, OCN and OSX were detected by RT-PCR. (E) The
expressions of osteogenic markers and Wnt/b-catenin/PI3K/Akt signaling determined by Western blot. **P < 0.01 and ***P < 0.005,
versus control.

808
 Journal of Dental Sciences 17 (2022) 802e810
substrate of PI3K/Akt signaling.47 PI3K/Akt signaling in-
hibits GSK3b by enhancing its phosphorylation at the Ser9
site. Our data suggested that inhibition of GSK3b activated
PI3K/Akt signaling, which was related to the role of AR in
promoting migration and osteogenic differentiation of
rDPSCs. Our data hinted a novel regulatory loop between
GSK3b and PI3K/Akt in rDPSC migration and osteogenic
differentiation.
In conclusion, the present study indicated that AR, a
GSK3b inhibitor, promoted migration and osteogenic dif-
ferentiation of rDPSCs in vitro. Activation of theb-catenin/
PI3K/Akt signaling pathway was involved in the effect of AR
on migration and osteogenic differentiation of rDPSCs.
Declaration of competing interest
The authors have no conflicts of interest relevant to this
article.
Acknowledgements
This work was supported by the Natural Science Foundation
of Fujian Province (grant number 2019J01503).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.jds.2021.09.035.
References
1. Kucharzewski M, Rojczyk E, Wilemska-Kucharzewska K, Wilk R,
Hudecki J, Los MJ. Novel trends in application of stem cells in
skin wound healing. Eur J Pharmacol 2019;843:307e15.
2. Mahla RS. Stem cells applications in regenerative medicine and
disease therapeutics. Int J Cell Biol 2016;2016:6940283.
3. Miana VV, González EAP. Adipose tissue stem cells in regener-
ative medicine. Ecancermedicalscience 2018;12:822.
4. Grove JE, Bruscia E, Krause DS. Plasticity of bone marrow-
derived stem cells. Stem Cell 2004;22:487e500.
5. Chen JC, Goldhamer DJ. Skeletal muscle stem cells. Reprod
Biol Endocrinol 2003;1:101.
6. Sunil PM, Manikandan R, Muthumurugan, Yoithapprabhunath TR,
Sivakumar M. Harvesting dental stem cells - overview. J Pharm
BioAllied Sci 2015;7:S384e6.
7. Seo BM, Miura M, Gronthos S, et al. Investigation of multipotent
postnatal stem cells from human periodontal ligament. Lancet
2004;364:149e55.
8. Yamada Y, Nakamura-Yamada S, Kusano K, Baba S. Clinical
potential and current progress of dental pulp stem cells for
various systemic diseases in regenerative medicine: a concise
review. Int J Mol Sci 2019;20:1132.
9. Yamada Y, Nakamura S, Ito K, et al. A feasibility of useful cell-
based therapy by bone regeneration with deciduous tooth stem
cells, dental pulp stem cells, or bone-marrow-derived mesen-
chymal stem cells for clinical study using tissue engineering
technology. Tissue Eng 2010;16:1891e900.
809
10. Yamada Y, Ito K, Nakamura S, Ueda M, Nagasaka T. Promising
cell-based therapy for bone regeneration using stem cells from
deciduous teeth, dental pulp, and bone marrow. Cell Trans-
plant 2011;20:1003e13.
11. Iwata T, Yamato M, Washio K, et al. Periodontal regeneration with
autologous periodontal ligament-derived cell sheets - a safety and
efficacy study in ten patients. Regen Ther 2018;9:38e44.
12. Su P, Tian Y, Yang C, et al. Mesenchymal stem cell migration
during bone formation and bone diseases therapy. Int J Mol Sci
2018;19:2343.
13. Carbone EJ, Jiang T, Nelson C, Henry N, Lo KW. Small molecule
delivery through nanofibrous scaffolds for musculoskeletal
regenerative engineering. Nanomedicine 2014;10:1691e9.
14. Shi A, Heinayati A, Bao D, et al. Small molecule inhibitor of
TGF-b signaling enables robust osteogenesis of autologous
GMSCs to successfully repair minipig severe maxillofacial bone
defects. Stem Cell Res Ther 2019;10:172.
15. AlMuraikhi N, Ali D, Alshanwani A, et al. Stem cell library screen
identified ruxolitinib as regulator of osteoblastic differentiation
of human skeletal stem cells. Stem Cell Res Ther 2018;9:319.
16. Beurel E, Grieco SF, Jope RS. Glycogen synthase kinase-3
(GSK3): regulation, actions, and diseases. Pharmacol Ther
2015;148:114e31.
17. Shang YC, Wang SH, Xiong F, et al. Wnt3a signaling promotes
proliferation, myogenic differentiation, and migration of rat
bone marrow mesenchymal stem cells. Acta Pharmacol Sin
2007;28:1761e74.
18. Wang Y, Zhang X, Shao J, Liu H, Liu X, Luo E. Adiponectin
regulates BMSC osteogenic differentiation and osteogenesis
through the Wnt/b-catenin pathway. Sci Rep 2017;7:3652.
19. Martinez A, Alonso M, Castro A, Pérez C, Moreno FJ. First non-
ATP competitive glycogen synthase kinase 3 beta (GSK-3beta)
inhibitors: thiadiazolidinones (TDZD) as potential drugs for the
treatment of Alzheimer’s disease. J Med Chem 2002;45:1292e9.
20. Roca C, Campillo NE. Glycogen synthase kinase 3 (GSK-3) in-
hibitors: a patent update (2016-2019). Expert Opin Ther Pat
2020;30:863e72.
21. Morales-Garcı́a JA, Susı́n C, Alonso-Gil S, et al. Glycogen synthase
kinase-3 inhibitors as potent therapeutic agents for the treatment
of Parkinson disease. ACS Chem Neurosci 2013;4:350e60.
22. Leclerc S, Garnier M, Hoessel R, et al. Indirubins inhibit
glycogen synthase kinase-3 beta and CDK5/p25, two protein
kinases involved in abnormal tau phosphorylation in Alz-
heimer’s disease. A property common to most cyclin-
dependent kinase inhibitors? J Biol Chem 2001;276:251e60.
23. Bhat R, Xue Y, Berg S, et al. Structural insights and biological
effects of glycogen synthase kinase 3-specific inhibitor AR-
A014418. J Biol Chem 2003;278:45937e45.
24. Shakoori A, Mai W, Miyashita K, et al. Inhibition of GSK-3 beta
activity attenuates proliferation of human colon cancer cells in
rodents. Cancer Sci 2007;98:1388e93.
25. Kunnimalaiyaan S, Gamblin TC, Kunnimalaiyaan M. Glycogen
synthase kinase-3 inhibitor AR-A014418 suppresses pancreatic
cancer cell growth via inhibition of GSK-3-mediated Notch1
expression. HPB 2015;17:770e6.
26. Alksne M, Simoliunas E, Kalvaityte M, et al. The effect of larger
than cell diameter polylactic acid surface patterns on osteo-
genic differentiation of rat dental pulp stem cells. J Biomed
Mater Res 2019;107:174e86.
27. Vandesompele J, De Preter K, Pattyn F, et al. Accurate
normalization of real-time quantitative RT-PCR data by geo-
metric averaging of multiple internal control genes. Genome
Biol 2002;3. Research0034.
 H. Xie, Y. Lin and F. Fang
28. Spyridopoulos T, Lambropoulou M, Pagonopoulou O, et al. Re-
generated nerve defects with a nerve conduit containing
dental pulp stem cells in pigs: an immunohistochemical and
electrophysiological evaluation. J Reconstr Microsurg 2015;31:
516e26.
29. Syed-Picard FN, Du Y, Lathrop KL, Mann MM, Funderburgh ML,
Funderburgh JL. Dental pulp stem cells: a new cellular
resource for corneal stromal regeneration. Stem Cells Transl
Med 2015;4:276e85.
30. Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal
human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc
Natl Acad Sci U S A 2000;97:13625e30.
31. Zhang M, Zhang P, Liu Y, Zhou Y. GSK3 inhibitor AR-A014418
promotes osteogenic differentiation of human adipose-
derived stem cells via ERK and mTORC2/Akt signaling
pathway. Biochem Biophys Res Commun 2017;490:182e8.
32. Shen S, Zhang Y, Zhang S, et al. 6-bromoindirubin-3’-oxime
promotes osteogenic differentiation of periodontal ligament
stem cells and facilitates bone regeneration in a mouse peri-
odontitis model. ACS Biomater Sci Eng 2021;7:232e41.
33. Huang X, Zhong L, Hendriks J, Post JN, Karperien M. The ef-
fects of the WNT-signaling modulators BIO and PKF118-310 on
the chondrogenic differentiation of human mesenchymal stem
cells. Int J Mol Sci 2018;19:561.
34. Komori T. Regulation of proliferation, differentiation and
functions of osteoblasts by Runx2. Int J Mol Sci 2019;20:1694.
35. Takahashi T. Overexpression of Runx2 and MKP-1 stimulates
transdifferentiation of 3T3-L1 preadipocytes into bone-forming
osteoblasts in vitro. Calcif Tissue Int 2011;88:336e47.
36. Liu Q, Li M, Wang S, Xiao Z, Xiong Y, Wang G. Recent advances
of osterix transcription factor in osteoblast differentiation and
bone formation. Front Cell Dev Biol 2020;8:601224.
37. Renn J, Winkler C. Osterix-mCherry transgenic medaka for
in vivo imaging of bone formation. Dev Dynam 2009;238:
241e8.
810
38. Majidinia M, Aghazadeh J, Jahanban-Esfahlani R, Yousefi B.
The roles of Wnt/b-catenin pathway in tissue development and
regenerative medicine. J Cell Physiol 2018;233:5598e612.
39. Carter YM, Kunnimalaiyaan S, Chen H, Gamblin TC,
Kunnimalaiyaan M. Specific glycogen synthase kinase-3 inhibi-
tion reduces neuroendocrine markers and suppresses neuro-
blastoma cell growth. Cancer Biol Ther 2014;15:510e5.
40. Yadav AK, Vashishta V, Joshi N, Taneja P, AR-A. Used against
GSK3beta downregulates expression of hnRNPA1 and SF2/ASF
splicing factors. J Oncol 2014;2014:695325. 014418.
41. Darshit BS, Ramanathan M. Activation of AKT1/GSK-3b/b-Cat-
enin-TRIM11/Survivin pathway by novel GSK-3b inhibitor pro-
motes neuron cell survival: study in differentiated SH-SY5Y
cells in OGD model. Mol Neurobiol 2016;53:6716e29.
42. Peltier J, O’Neill A, Schaffer DV. PI3K/Akt and CREB regulate
adult neural hippocampal progenitor proliferation and differ-
entiation. Dev Neurobiol 2007;67:1348e61.
43. Yu JS, Cui W. Proliferation, survival and metabolism: the role
of PI3K/AKT/mTOR signalling in pluripotency and cell fate
determination. Development 2016;143:3050e60.
44. Lu SY, Wang CY, Jin Y, et al. The osteogenesis-promoting ef-
fects of alpha-lipoic acid against glucocorticoid-induced oste-
oporosis through the NOX4, NF-kappaB, JNK and PI3K/AKT
pathways. Sci Rep 2017;7:3331.
45. Yuan H, Zhao H, Wang J, et al. MicroRNA let-7c-5p promotes
osteogenic differentiation of dental pulp stem cells by inhib-
iting lipopolysaccharide-induced inflammation via HMGA2/-
PI3K/Akt signal blockade. Clin Exp Pharmacol Physiol 2019;46:
389e97.
46. Li M, Sun X, Ma L, et al. SDF-1/CXCR4 axis induces human
dental pulp stem cell migration through FAK/PI3K/Akt and
GSK3b/b-catenin pathways. Sci Rep 2017;7:40161.
47. Yang K, Chen Z, Gao J, et al. The key roles of GSK-3b in
regulating mitochondrial activity. Cell Physiol Biochem 2017;
44:1445e59.